Conventional wide field microscopy is based on formation of a high-magnification image of an illuminated sample using conventional microscope optics. In contrast, confocal microscopy is based upon illumination of a small part of the sample, referred to as a target region, and on selective collection of light emitted from the target region. Image formation is accomplished by scanning the position of the target region within the sample. Typically, the sample is illuminated with an illumination beam which is brought to a diffraction-limited (or nearly so) focus within the sample. Light emitted by the part of the sample within the focal region of the illumination beam is selectively collected and detected.
It is helpful to define an observation beam as being the beam that would be present if the optical detector in the above selective collection and detection arrangement were replaced by an optical source. Parts of the sample outside the observation beam are generally “not seen” by the detector. Thus the overlap of the illumination beam and observation beam defines the target region. Since it is generally desirable to decrease the size of the target region as much as possible, the illumination beam and observation beam are typically both brought to a small diffraction-limited focus (e.g., using a high numerical aperture (NA) lens having low aberration). Furthermore, the focal regions of the illumination beam and observation beam typically overlap (i.e., the two beams are typically confocal).
In the earliest confocal microscopes, the illumination beam and observation beams are collinear. In fact, frequently the same optical elements define the observation and illumination beams, and the observed signal is separated from the illumination light with a beamsplitter or directional coupler. When a beam is brought to a focus, the resulting focal region typically has an axial dimension several times larger than its transverse dimensions, especially if the focusing numerical aperture is less than 0.5. Here the axial direction is along the beam axis and the transverse directions are perpendicular to the beam axis. Thus, collinear illumination and observation beams typically provide a generally “cigar shaped” target region, having an axial dimension several times larger than its transverse dimensions.
More recently, for example in U.S. Pat. No. 5,973,828, non-collinear illumination and observation beams have been employed. Since the two beams intersect at an angle, the resulting target region is smaller than it would be for collinear beams. In particular, the target region can be roughly spherical and can have a radius on the order of the transverse beam dimensions. Such confocal microscopes are referred to as dual axis confocal microscopes.
A further variant of a dual axis confocal microscope is considered in U.S. Pat. No. 6,369,928, where two non-collinear illumination beams are supplied to the sample. In this arrangement, the illumination beam optics can conveniently define non-collinear observation beams (e.g., illumination optics 1 defines observation beam 2 and vice versa). Alternatively, light emitted from a sample region where the two illumination beams overlap can be selectively collected by optics other than the illumination beam optics.
In some cases, it is desirable to perform dual axis confocal microscopy on a sample having a significant thickness, such that the target region is within the sample as opposed to being on a sample surface. For example, biological or medical applications of confocal microscopy frequently require the ability to image structures within a tissue sample.
However, significant beam aberration can occur when a beam is non-normally incident on an interface having a refractive index discontinuity. Since a thick sample typically entails at least one such interface, this source of aberration must be considered in dual axis confocal microscopy of thick samples. One approach for alleviating this difficulty is considered in an article by Wang et al. in Optical Letters 28(2) pp 1915–7 2003, where the sample is tissue, and beams pass through a prism, a water bead, and a cover glass before reaching the sample. The prism and water have an index close to that of the tissue sample, and the beams are normally incident on the prism-air interfaces. But the approach of Wang et al. is complex (since many optical elements are required) and inflexible (since it is not straightforward to add additional input or output beams).
Accordingly, it would be an advance in the art to provide a dual axis confocal microscope for use with thick samples having a simpler and more flexible configuration than previously known.